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MICROSCOPY RESEARCH AND TECHNIQUE 40:488–491 (1998)
Immunocytochemical Localization of Adenylyl Cyclase
in Human Myometrium
PETER D.G. RICHARDS,1* ANDREW J. TILTMAN,1 AND PENELOPE A. RICHARDS2
1Department
2Department
of Anatomical Pathology, SAIMR, Johannesburg, Republic of South Africa
of Anatomy, University of Pretoria, Pretoria, Republic of South Africa
KEY WORDS
adenylyl cyclase V/VI; smooth muscle; relaxation
ABSTRACT
The enzyme adenylyl cyclase (AC) plays a pivotal role in smooth muscle relaxation.
Biochemical evidence suggests that AC is predominantly located in the outer layers of the
myometrium; however, neither immunocytochemical nor histochemical studies have been undertaken to demonstrate the specific cellular distribution of the enzyme in this tissue. As part of an
ongoing study of the human myometrium, a polyclonal antibody against types V and VI AC was used
to detect the presence of these isoforms in sections of formalin-fixed, wax-embedded myometrial
tissue. A positive reaction was seen in the cytoplasm of the smooth muscle cells with the
midmyometrial area having the greatest number of positive cells, when compared to the subserosal
and subendometrial areas. It is hypothesized that AC isoform type VI is the predominant isoform
present in the myometrium and that the percentage distribution of positive cells reflects the area of
highest myometrial activity during parturition. Microsc. Res. Tech. 40:488–491, 1998.
r 1998 Wiley-Liss, Inc.
INTRODUCTION
The human myometrium consists of interlacing
bundles of smooth muscle in a loose connective tissue
matrix. The myometrial wall itself can be histologically
divided into three layers of variable distinction: the
inner subendometrium, the midmyometrium, and the
outer subserosal layer. Recent immunocytochemical
studies have elucidated that estrogen receptors are not
uniformly distributed through the depth of the myometrial wall (Richards and Tiltman, 1995). Similarly,
biochemical evidence suggests that adenylyl cyclase
(AC), an enzyme, is also differentially distributed in the
myometrium (Fortier and Krall, 1983); however, this
distribution does not appear to have been studied by
either histochemical or immunocytochemical means
and, thus, the exact cellular localization of AC remains
unknown. AC is a membrane-bound enzyme that cyclisizes adenosine triphosphate (ATP) to cyclic adenosine
monophosphate (cAMP). In the context of smooth
muscle, agents that induce relaxation, such as βadrenergic agonists, act directly through the AC-cAMP
system (Lincoln and Cornwell, 1991).
Since the elucidation of the structure and amino acid
sequence of AC by Krupinski et al. (1989), at least eight
isoforms have been cloned (Watson and Arkinstall,
1994). In situ hybridization studies and Western blot
analysis using the cloned isoforms have determined
their distribution in a number of tissues. Types I, II, III,
VII, and VIII are usually located in mammalian neural
tissue (Bakalyar and Reed, 1990; Cali et al., 1994;
Cooper et al., 1995; Feinstein et al., 1991; Krupinski et
al., 1989;), while types IV, V, and VI have a wider
distribution in tissues such as heart, liver, and lung
(Gao and Gilman, 1991; Ishikawa et al., 1992; Premont
et al., 1992). Unfortunately, there has been little indepth immunocytochemical analyses of any of these
tissues (Mons et al., 1995), with few studies having
r 1998 WILEY-LISS, INC.
screened uterine tissue in order to ascertain the isoform
present. As both in situ hybridization and Western blot
analysis have shown isoform type I to be absent from
uterine tissue (Xia et al., 1993), it would be unlikely for
the other predominantly neurally expressed isoforms to
be present in myometrial cells. Similarly, it is questionable whether type IV occurs in the myometrium, for
although it is present in human tissue, its occurrence is
rare (Cooper et al., 1995). Therefore, as part of a wider
study of the human myometrium, a polyclonal antibody
against the C-terminal amino acids of the more widely
distributed human isoforms, types V and VI, was used
to determine 1) whether these isoforms are present in
myometrial tissue and 2) if present, what the distribution of AC is within the myometrial wall.
MATERIALS AND METHODS
Samples of uterine tissue were obtained from eight
patients undergoing hysterectomy for either menorrhagia or dysmenorrhoea. The patients ranged in age from
34 to 56 years and had no history of exogenous hormone
treatment. Uteri were opened in the sagittal plane
within 10 minutes of surgical excision from the patients. A transmural block of tissue was dissected from
the fundus of each uterus, with both the endometrium
and serosa intact. Following fixation in 10% formalin,
the tissue was processed for routine histology. Wax
sections stained with Mayer’s haematoxylin and eosin
were used to determine the normality of the tissue and
the phase of the endometrial cycle.
Contract grant sponsor: Medical Research Council of South Africa.
*Correspondence to: P.D.G. Richards, Department of Anatomy, Faculty of
Medicine, University of Pretoria, PO Box 2034, Pretoria 0001 Republic of South
Africa.
Received 10 September 1996; accepted in revised form 6 January 1997.
ADENYLYL CYCLASE IN MYOMETRIUM
The presence of AC types V and/or VI was immunocytochemically determined using a streptavidin-biotin
technique. Briefly, sections of 4 µm thick were dewaxed
and placed in a 3% methanolic solution of hydrogen
peroxide for 30 minutes at room temperature in order to
block endogenous peroxidase activity. Secondary antibody binding sites were blocked by placing the sections
in a solution of 5% normal horse serum for 30 minutes
at room temperature. The sections were then incubated
with the primary antibody, a polyclonal rabbit antibody
against AC V and VI (2 µg/ml) (Santa Cruz Biotech,
USA), for 60 minutes at room temperature. A biotinylated horse anti-rabbit (1:200) secondary antibody (Vecta,
USA) was applied for 30 minutes, following which the
sections were treated with a streptavidin-peroxidase
conjugate kit (Vecta, USA) for a period of 45 minutes.
The peroxidase was visualized using a diaminobenzidine (DAB)-hydrogen peroxide solution (0.1 g/100 ml) (5
minutes at room temperature) enhanced with cobalt
chloride. Each application of antibody was followed by
three changes of phosphate buffer with 0.5% bovine
serum albumin (pH 7.2). This buffer was also used for
the dilution of the antibodies. Liver sections known to
contain AC V and VI (Watson and Arkinstall, 1994)
were used as positive controls and processed along with
the myometrial sections. Negative control sections were
processed in exactly the same manner, except that they
were placed in normal horse serum instead of the
primary antibody against AC V and VI. All the sections
were counterstained with Mayer’s haematoxylin, dehydrated, and mounted. Sections were viewed and photographed using bright-field microscopy.
For counting purposes, the myometrial sections were
subdivided into three regions of equal width; these
being the subendometrium, midmyometrium, and subserosa. The total cell population of 10 oil immersion
fields (0.28 mm2) was counted in each region per slide,
where a cell was counted as positive when it showed
evidence of DAB reaction product in the cytoplasm or
nucleus.
RESULTS
Examination of the haematoxylin and eosin-stained
sections confirmed the histological normality of each
specimen. All phases of the endometrial cycle were
represented by the selected myometrial samples (secretory n 5 4; proliferative n 5 3; menstrual n 5 1). The
DAB reaction product, associated with all positive
myometrial cells, usually obscured the entire cytoplasmic component, although sporadic nuclei also stained
positive. Staining intensity was inconsistent, with some
fibres showing distinctive reaction product while others
were only lightly stained (Fig. 1). Mast cells and
vascular smooth muscle cells were occasionally positive
(Fig. 2). Control sections of liver that had not been
incubated in the primary antibody were negative (Fig.
3a), while sections incubated in the presence of the
primary antibody acted as positive controls (Fig. 3b).
Table 1 shows the average number of cells counted
per high-power field, as well as the percentage positivity for each of the three regions. The midmyometrial
region demonstrates the highest percentage positivity
when compared with the subserosal and subendometrial regions. Despite the differences in the number of
cells between the three regions, the subserosal and
489
Fig. 1. Light micrograph of the midmyometrial region stained with
an antibody against type V and VI AC. There is a large number of
positive cells present with some staining darker (arrows) than others.
Scale bar 5 100 µm.
Fig. 2. Light micrograph of the midmyometrial area stained with
an antibody against type V and VI AC. The prominent blood vessel is
strongly positive (arrows). Scale bar 5 100 µm.
subendometrium are both significantly less positive
than the midmyometrium (analysis of variance P ,
0.0001). There is no significant difference in positivity
between the subserosal and subendometrial areas,
although the latter is the least positive. Age appears to
be an influencing factor in the positivity of the midmyometrium, where there is a significant decrease in
positivity with advancing age (analysis of variance P ,
0.001). No statistically significant relationship was
demonstrated between the phase of the endometrial
cycle and the positivity of the specific regions of the
myometrium.
DISCUSSION
The positive reaction obtained with the AC V/VI
antibody, indicates the presence of AC isoform type V
and/or VI in human myometrial cells. As these two
isoforms are 93% similar with respect to the amino
490
P.D.G. RICHARDS ET AL.
Fig. 3. Light micrograph of a section of liver. (a) Negative control
and (b) positive control. Scale bar 5 100 µm.
TABLE 1. The average number of positive cells counted per high
power field (HPF) and the percentage positivity for each region of the
myometrium irrespective of the stage of the endometrial cycle
Average 1ve cells per HPF
% positivity
SE
(n 5 80)
MM
(n 5 80)
SS
(n 5 80)
108
27.10
84
69.74*
56
56.57
*Significantly higher percentage positivity in the midmyometrial (MM) region
when compared to the subendometrial (SE) and subserosal (SS) regions.
acids at the C-terminal end (Watson and Arkinstall,
1994) it is not possible, using this antibody, to determine which of the two isoforms predominates. In terms
of their specific distribution in tissues of muscular
origin, type V occurs only in cardiac muscle, while type
VI has been isolated in both cardiac and smooth muscle
(Watson and Arkinstall, 1994). Considering that the
most abundant source of mRNA for AC type VI is
cardiac muscle, while for all of the other isoforms it is
from tissues of neural origin (Cooper et al., 1995), it is
probable that the AC isoform present in myometrial
tissue is type VI.
Even though the specific AC isoform in myometrial
tissue is not known, its pivotal role in the relaxation of
smooth muscle by β-adrenergic agonists has been established (Carsten and Miller, 1987). Smooth muscle contraction and relaxation are determined by the intracellular calcium ion concentration ([Ca21]I) (Somolyo and
Somolyo, 1994), where initiation of contraction results
from an increase in [Ca21]I, due to the release of Ca21
from intracellular stores. Relaxation is the consequence
of a lowering of [Ca21]I due to the activation of plasmaand sarcolemmal Ca21 adenosine triphosphatase (Ca21
ATPase) pumps. Relaxation is induced by agents such
as β-adrenergic agonists which activate AC, thus increasing the levels of cAMP in the cell. The rise in cAMP
levels in turn activates protein kinase A and cyclic
guanosine monophosphate kinase, these being two
enzymes that activate the Ca21 ATPase pumps (Lincoln
and Cornwell, 1991).
In the nongravid uterus, myometrial activity increases through the menstrual cycle to a peak in the
late secretory period (Bell et al., 1968). In the gravid
uterus, myometrial contractions start in earnest from
the 20th week with an increasing frequency towards
term, with total contraction of the uterus taking place
at the end of the third stage of labour (Bell et al., 1968).
The percentage distribution of the AC protein as revealed by this study implicates the midmyometrial
region as being the most sensitive to relaxation stimuli
and, thus, probably the area of the myometrium with
the highest work potential during periods of contraction. The sustained contraction of the uterus at the end
of the third stage of labour is probably achieved by the
inhibition of AC (types V and VI) following the increase
in [Ca21]I that results from exogenous Ca21 influx via L
type channels (Yu et al., 1993).
The myometrium changes as it ages, atrophying after
the climacteric as a result of the loss of the bulk and
tone, which are normally maintained by estrogen (Llewllyn-Jones, 1982). If AC positivity reflects areas of high
muscular activity, then any loss in such activity as a
result of aging or disease should be reflected by a
decrease in AC positivity. The decrease in AC positivity
seen with increasing age in this study is probably due to
decreasing estrogen stimulation and myometrial bulk
in the midmyometrial region with advancing age.
In conclusion, it is probable that AC isoform type VI
is present in myometrial tissue, its main distribution in
the myometrium being in the midmyometrial area of
the muscle, where it probably plays a major role in the
relaxation of myometrial smooth muscle during parturition.
ACKNOWLEDGMENTS
The authors wish to acknowledge the technical assistance of Ms. Z. Haffejee and Ms. L. Taylor as well as Dr.
A. Bener for his assistance with the statistical analysis.
This work forms part of a MSc thesis being undertaken
at the University of the Witwatersrand, Republic of
South Africa.
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